Gas phase biocatalysis method and process

ABSTRACT

A method of enzyme conversion comprises the steps of:
         immobilising an enzyme composition on a support material;   drying the support material and enzyme composition to form a solid phase immobilised enzyme system;   contacting the system with one or more reagents in the gas phase;   allowing the enzyme system to convert the reagent(s) to product(s);   wherein the enzyme composition may comprise a single enzyme or a first enzyme plus a second enzyme or multiple enzymes; and a co-factor which may be converted between first and second states;   wherein the co-factor in the first state promotes reaction of the first enzyme; and   wherein the co-factor in the second state promotes reaction of the second enzyme;   or wherein the co-factor oscillates between first and second states with multiple enzymes.

This invention relates to a method of enzymic conversion in which anenzyme composition is mobilised on a solid substrate for reaction with agaseous reagent. The invention also relates to an enzymic conversionsystem for use in the method.

A number of processes describing biocatalytic reactions in gas phaseapplications have been described. However they have differentrequirements which make them partially ‘gas phase’ systems. U.S. Pat.No. 5,670,332 (Kuhl et al, Degussa) discloses solid phase systemsoperating by gas phase reactants being passed through a mixture ofenzyme and solid phase substrates. The system is able to convert thesolid phase substrate into a product in the mixture which issubsequently extracted to recover the product. Examples given includepeptides generated using proteases such as chymotrypsin.

U.S. Pat. No. 6,440,711 Bakul Davé, (Southern University of Illinois)discloses a sol-gel immobilised enzyme system comprising formatedehydrogenase, formaldehyde dehydrogenase and alcohol dehydrogenaseconfigured into the pores of the sol-gel to generate methanol from CO₂and reduced co-factor NADH. In this case liquid water is added and theCO₂ is bubbled through the suspension. Photosystem II (PSII) obtainedfrom spinach leaves was added to NADH to enable the continuousproduction of methanol to occur in solution. The sol-gel system isimportant to make the enzymes function effectively in the reversedirection. Publications by Kuwabata et al., (1994), Kuwabata et al.,1990 and Mandler et al., (1988) are cited as supporting documentation.

WO2011/163323 discloses a modified carbonic anhydrase enzyme that isimmobilised into a reactor and which is used in a process to remove CO₂from an atmosphere. However a first step requires the CO₂ to be capturedinto a liquid medium, e.g. water. The water/CO₂ mixture is then passedover the immobilised modified enzyme to give bicarbonate ions which arethen removed by a metal precipitation step using calcium ions. Thisresults in overall removal of CO₂ from the atmosphere. This is the basisof a commercial process by CO₂ Solutions Inc. However the use of wateras a solvent for CO₂ will introduce mass transfer barriers in theprocess. Also the CO₂ ends up as a solid precipitate of calciumcarbonate and not a useful fine chemical such as formic acid.

SUMMARY

According to a first aspect of the present invention, a method ofenzymic conversion comprises the steps of:

immobilising an enzyme composition on a support material;

drying the support material and enzyme composition to form a solid phaseimmobilised enzyme system;

contacting the system with a gaseous reagent; and

allowing the enzyme system to convert the reagent to a product;

wherein the enzyme composition comprises a first enzyme, a second enzymeand a co-factor which may be converted between first and second states;

wherein the co-factor in the first state promotes reaction of the firstenzyme; and

wherein the co-factor in the second state promotes reaction of thesecond enzyme.

According to a second aspect of the present invention, an enzymicconversion system comprises:

a dried enzyme composition on a support material comprising a firstenzyme, a second enzyme and a co-factor which may be converted betweenfirst and second states;

wherein the co-factor in the first state promotes reaction of the firstenzyme; and

wherein the co-factor in the second state promotes reaction of thesecond enzyme.

The first and second states of the co-factor may be oxidized or reducedstates. A preferred co-factor is NAD/NADH.

The enzyme is preferably dried to a sufficient extent to produce animmobilised enzyme system. However, the enzyme is preferably not driedto such a low water level that it is deactivated. A water content of0.05% to 5% w/v may be employed.

Use of the method of the present invention allows complex multi-subunitenzymes to operate catalytically in gas phase systems.

The support may be an inorganic substrate, for example sand, silica orglass. The support material may be provided in the form of particles orbeads. Preferably, porous beads are employed. These may have a highinternal surface area.

The system may comprise a fluidised bed in which the particulate supportmaterial is fluidised by passage of the gaseous reagent.

The first enzyme may be selected from the group consisting of methanemono-oxygenase (MMO) and alkene mono-oxygenase (AMO) or any othermono-oxygenase.

The second enzyme may be selected from one or more dehydrogenaseenzymes, for example:

alcohol dehydrogenase;

formaldehyde dehydrogenase; or

formate dehydrogenase.

In preferred embodiments of the invention, liquid water is absent fromthe enzyme composition, reagent and the support.

In preferred embodiments, the enzymes are dried on the support.Immobilisation of the enzyme within a sol or gel is not preferred.

Use of a silica or other solid substrate supported enzyme system isadvantageous to improve the mass transfer properties of the process, aswater is not used. Diffusion processes are much faster in the gas phasethan in the liquid phase, leading to higher rates of reaction.

Furthermore, use of a gas phase system confers a considerable increasein operating longevity or thermostability of the enzymes employed.

Use of a system including MMO as an enzyme component provides a methodfor conversion of methane using molecular oxygen to form methanol. Thisprovides a process for production of methanol using the cheap substratesmethane and oxygen.

In a multi-enzyme system, alcohol dehydrogenase (ADH), formaldehydedehydrogenase (FADH) and formate dehydrogenase (FODH) may be immobilisedon a solid support to produce formic acid from methanol in a gas phasereaction.

In a further multi-enzyme system, methane mono-oxygenase (MMO), alcoholdehydrogenase (ADH), formaldehyde dehydrogenase (FADH) and formatedehydrogenase (FODH) may be immobilised on a support to produce formicacid from methane, oxygen and carbon-dioxide. The enzymes may beimmobilised as a mixture or may be located on individual support regionsof the supports.

Use of the enzyme alkene mono-oxygenase allows the conversion of analkene such as propylene to a chiral product such as R-propylene oxide.The enzyme reaction significantly favours the production of one chiralform over the other.

A multi-enzyme system comprising alkene mono-oxygenase (AMO), alcoholdehydrogenase (ADH), formaldehyde dehydrogenase (FADH) and formatedehydrogenase (FODH) may be immobilised on a support in order to produceCO₂ and water from methanol and R-propylene oxide from propylene usingNADH recycled from NAD via the oxidation of methanol.

The system of this invention may be used as a sensor to detect thereagent in a dry phase test.

Enzymes when immobilised in a low water environment may retainsignificant biocatalytic activity, provided there is a micro-environmentthat maintains a certain amount of water around the enzyme structure.The invention disclosed combines production of a stable admix of enzymesand cheap, simple support materials such as porous silica particles, arapid vacuum assisted drying step at elevated temperature and thedemonstration of enzyme biocatalytic activity with purely gaseous and/orvapour phase substrates to generate fine chemical products.

It was unexpected that an extremely complex multi-subunit enzyme,methane mono-oxygenase (MMO), was found to retain biocatalytic activitywhen immobilised allowing conversion of gaseous methane to methanolusing indigenous reduced co-factor NADH and oxygen in the gas phase.Unpurified, crude preparations of MMO may be used to biocatalyticallygenerate methanol from methane. The reduced co-factor NADH may berecycled within a silica supported enzyme mixture, purely with gas phasereactions, i.e. no liquid water added is also an important feature.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described by means of example but not in anylimitative sense with reference to the accompanying drawings, of which:

FIG. 1 shows a reaction sequence in accordance with the invention;

FIG. 2 shows a reaction sequence for alcohol dehydrogenase biocatalysis;

FIG. 3 shows a methanol calibration plot;

FIG. 4 shows a dry phase methanol detection device;

FIG. 5 shows a gas chromatograph for acetone conversion to isopropanoland prenol to prenal;

FIG. 6 illustrates conversion of methanol to formic acid;

FIG. 7 shows a reaction sequence for alkene mono-oxygenase biocatalysis;and

FIG. 8 shows a R-propylene oxide calibration plot.

DETAILED DESCRIPTION

To demonstrate the recycling of the NAD/NADH co-factor couple acombination of dehydrogenase enzymes, alcohol dehydrogenase,formaldehyde dehydrogenase and formate dehydrogenase were used in thesequence shown in FIG. 1.

A major advantage of the present process is that an enzymatic conversionmay be carried out in the gas phase, with the enzyme supported in asolid dry state on porous beads with a high internal surface area. Thisform of supported enzyme material is suitable for fluidisation where thereactant(s) in the gas phase lifts the enzyme beads creating aparticulate fluid form. The effect of this is to greatly enhance themass transfer properties of the process and allow much more efficientbiocatalysis. No liquid water being present, the access of thesubstrates to the active sites of the enzymes is not prevented by poormass transfer.

A second advantage is that substrates that are water insoluble but whichhave a vapour pressure, i.e. which are volatile can reach the enzymeswith minimal issues with the water insolubility. Methane is a goodexample of this as it is very sparingly soluble in water but it isconverted to methanol by the MMO in the gas phase quite efficiently.

Single Enzyme Systems

1) Methane Mono-Oxygenase

A single enzyme that transforms methane using molecular oxygen in thepresence of reduced co-factor NADH was tested. Methane mono-oxygenase(MMO) is a multi-subunit enzyme with three distinct components:

a hydroxylase sub-unit that reacts with methane to make methanol;

a reductase sub-unit acts to make NADH and supply the reducing power tothe hydroxylase;

protein B acts as a regulation sub-unit and promotes hydroxylaseactivity.

All 3 sub-units need to be present and active to make MMO workcorrectly.

One millilitre of crude MMO lysate was used to produce an immobilisedMMO preparation. This was tested for activity by blowing a mixture ofmethane and air and CO₂ through an immobilised substrate and theresulting absorbance at a wavelength of 500 nm was measured. See Example1 below.

2) Alcohol Dehydrogenase

A second single enzyme system based on yeast alcohol dehydrogenase (ADH)was immobilised on silica beads.

The appearance of prenal and isopropanol in the exhaust gas stream wasobserved using a gas chromatography method, indicating enzyme activitywas occurring according to FIG. 2.

Multienzyme Systems

1) Alcohol dehydrogenase (ADH) formaldehyde dehydrogenase (FADH) andformate dehydrogenase (FODH) were coupled together to produce formicacid from methanol in a gas phase reaction seen in FIG. 1. This reactionsequence was used to generate useful fine chemicals from cheaperprecursors and at the same time provide a CO₂ sequestration system thatwould fix the CO₂ into an organic fine chemical, suitable for use inchemical synthesis.

The overall reaction sequence was:CH₃OH+H₂O+2CO₂=3HCOOH.

2) A second multienzyme system based on four enzymes was used. Methanemono-oxygenase (MMO), alcohol dehydrogenase (ADH) formaldehydedehydrogenase (FADH) and formate dehydrogenase (FODH) were coupledtogether to produce formic acid from methane, oxygen and CO₂. Theoverall reaction performed by the enzyme system was as follows:CH₄+CO₂+O₂=2HCOOH.

In this case various mixtures of the dry enzymes ADH, FADH and FODH wereadded to 1 ml of sMMO crude lysate together with 1 ml of 50 mM sodiumphosphate buffer pH 7.5, dissolved completely and then added to 200 g ofsilica beads. Dehydration was carried out and the resulting dry powderpacked into a 5 cm column. Introduction of methane, air and CO₂ resultedin the production of formic acid which was measured quantitatively usinga colorimetric formazan assay as before. See Example 4.

3) A third multi-enzyme system based on four enzymes was used. Theenzymes alkene mono-oxygenase (AMO), alcohol dehydrogenase (ADH),formaldehyde dehydrogenase (FADH) and formate dehydrogenase (FODH) werecoupled together to produce CO₂ and water from methanol and R-propyleneoxide from propylene.

The reaction catalysed by AMO is as follows:

The reaction catalysed by the three enzymes used to regenerate thecofactor (ADH, FADH and FODH) is:CH₃OH+3NAD⁺+H₂O=3NADH+3H⁺+CO₂

Combining the two reactions yields the net reaction below:

See Example 5.

EXAMPLES

1. Conversion of Methane to Methanol

Methane is fixed by various methylotrophic bacteria and the enzyme thatdoes this is Methane Mono-Oxygenase (MMO). This is a multi-subunitenzyme.

A crude cell lysate produced from a cell paste harvested from thefermentation of the methanotrophic bacteria, Methylosinus trichosporiumOB3b (Fox et al. J Biol Chem 264 (1969) p 10023-10033) was used toproduce the soluble MMO (sMMO).

The crude lysate contained approximately 3 mg per ml of sMMO and to 200mg of silica beads (SI 1410 grade, Grace Davidson Catalysts), 1 ml oflysate was added. The mixture was dehydrated over a 2 hour period underreduced pressure over a bed of freshly dried silica gel (2-3 kg inweight).

The resulting dry powder was stored at 4° C. until required.

The powder was weighed into a 5 cm length of 6 mm (¼ inch)polyfluoroacetate tubing with porous polystyrene plugs at each end toproduce a mini-column reactor that allowed unrestricted gas flow.Typically 150-200 mg powder was used.

The gas flow used was made up of a mixture of methane (4-5 ml perminute, air 10-12 ml per minute and sometimes CO₂ was added at 4-5 mlper minute to see if this had any effect on the catalytic rate).Methanol production was measured using a colorimetric reaction based onalcohol oxidase, horseradish peroxidase and colour reagents4-aminoantipyrine and N, N′-bis hydroxyethyl aniline, which gives apurple imino dye on reaction with methanol.

Removal of methane from the gas mixture resulted in no colour, whereasaddition of methane gave a strong purple colour indicating activebioconversion

Colour development was checked against a calibration plot for methanol(FIG. 3 below) and found to give almost 1 mMole of methanol in 20minutes incubation at room temperature.

Sample Absorbance Methanol (μM) Methanol production air and methane0.209 946Rapid Enzyme Paper Method for Detection of Methanol.

A visual dry phase test was used. This allowed the detection of methanolon a solid dry phase support, as shown in FIG. 4. FIG. 4 shows anannular support (1) carrying a disc (2) impregnated with enzyme andcolour magnets and several by a porous circlip (3).

Analyte containing gas (4) from a reactor (not shown) passes through thediffuser to create a visible colour reaction in the enzyme disc.

The off gases from the enzyme reactor caused a purple spot in the centreof the enzyme paper when methanol was present. This is not quantitativebut as a qualitative test it is very useful to visualize methanolproduction in a few minutes. This is shown in FIG. 5 a.

The methanol produced was due to MMO activity and not just an artifactor impurities in reagents. With just air and CO₂ alone for 1 hour, thentesting the off gases with the rapid paper method gave no visual colour,indicating no methanol was present in the off gases. Adding methane intothe gas stream and incubating for 1 hour again, then testing with thepaper detector gave a strong purple colour in the centre of thedetector. Indicating methanol was being produced again from the methaneadded by the MMO. This is shown in FIG. 5b

2. Alcohol Dehydrogenase

Bovine serum albumin (2 g) was added to 10 ml of 50 mM phosphate bufferpH 7.8 and added to 4 g of SI 1410 silica beads. The mixture was driedovernight at 40° C. under reduced pressure and sieved through a 125micron metal sieve to give free flowing BSA-silica powder. Freeze driedyeast alcohol dehydrogenase (2 g) was added 10 ml of 50 mM phosphatebuffer pH 7.8, plus 1 ml of 15 mM NAD in the same buffer and added to 4g of the pre-prepared BSA-silica beads. The mixture was dried overnightat 40° C. under reduced pressure and sieved through a 125 micron metalsieve to give free flowing ADH-BSA-silica powder.

The ADH-BSA-silica bead preparation (1 to 2 g) was packed into a 1 cminternal diameter tube to give a fixed bed column that was then perfusedwith gas phase acetone and prenol vapours in air at 4 ml per minute overa period of several hours (usually over 8 hours).

The appearance of prenal and isopropanol in the exhaust gas stream wasobserved using a gas chromatography method (GC), indicating enzymeactivity was occurring according to FIG. 6.

Further to this method, which is slow to produce product, a fluidisedbed was tested. Here 15 g BSA-silica (1 g to 1 g ratio) was added to 15g of BSA-ADH-silica (400 mg BSA plus 500 mg ADH per gram silica) wasadded to a 2 inch column reactor and gas phase acetone and prenolvapours in air was introduced at 12 ml per minute to give an airsupported fluidised bed of enzyme beads. The reactor produced a 1:1ratio of isopropanol/acetone for several days indicating enzymaticconversion was taking place continually in the dry phase.

3. Conversion of Methanol to Formic Acid with Concomitant CO₂Sequestration

A preparation of freshly immobilised enzyme beads made with 161 mg ADH,152 mg FADH and 199 mg FODH added to 9 ml of 50 mM phosphate buffer pH7.5 plus 1 ml of 15 mM NAD. This was added to 1.032 g BSA coated silicabeads and then vacuum dried overnight at 40° C.

The resulting dry powder was then gently ground and passed through a 125μm sieve to remove any larger aggregates. The large aggregates werere-ground and sieved until the whole volume of the enzyme beads were 125μm or smaller.

These dry enzyme beads were then fluidised in the gas phase rig usingCO₂ and methanol was introduced into the flowing stream to start thereaction. The off-gasses were captured in 1.5 ml 10 mM NaOH solution andassayed using an enzymatic formate assay based on the production of ared formazan from the NADH generated by formate dehydrogenase and NAD.This reaction was absolutely specific for formic acid and formate salts.Therefore any colour development is the result of the presence of theseentities in solution.

The reaction was run for 2 hours initially and the results are shown inFIG. 7.

The duplicate assays are shown plus the average of the two. In this case120.4 μMolar formic acid was measured in the 1.5 ml reaction volume,indicating that the three enzyme system of ADH/FADH/FODH has usedmethanol and CO₂ present in the gas phase with the added NAD, enablingcofactor re-cycling to produce formic acid. In all cases the carrier gas(CO₂) was humidified with water before it was passed over the threeenzyme beads, leading to the likely formation of a water film on thebead surfaces. In real terms at the nanoscale there would be asubstantial layer of water present and the NAD would be dissolved inthis layer, allowing to cofactor migration between the enzymes and thuscofactor recycling between the three enzymes in this system. This mayreflect the natural conditions for enzyme activity, since in cells thewater activity is much lower than in vitro experiments, i.e. there isnot much liquid water available under normal metabolic conditions. Incells, enzymes do not work in dilute solutions but are present in highlyconcentrated states.

4. Conversion of Methane to Formic Acid with Concomitant CO₂Sequestration

A preparation of freshly immobilised enzyme beads made with 150 mg ADH,150 mg FADH and 200 mg FODH added to 8 ml of 50 mM phosphate buffer pH7.5 plus 1 ml of 15 mM NAD and 1 ml of crude MMO lysate. This was addedto 1.0 g BSA coated silica beads and then vacuum dried overnight at 40°C.

The resulting dry powder was gently ground and passed through a 125 μmsieve to remove any larger aggregates. The large aggregates werere-ground and sieved until the whole volume of the enzyme beads were 125μm or smaller.

The mini-column reactor was constructed as before using between 200-250mg of enzyme beads in the perfluoroacetate column. Different mixtures ofgases were perfused through the beads and the formic acid generated wasmeasured using the specific formate formazan assay described previously.

Using just air and methane generated formic acid from the MMO, ADH andFADH.

Adding CO₂ to the gas mixture adds the FODH reaction and an increase inthe formic acid produced. This is shown in Table 1 overleaf.

TABLE 1 Generation of Formic Acid from MMO, ADH, FADH and FODH Beads.Formic Acid Average (μM.mg⁻¹ on Sample Time Absorbance beads DistilledWater Blank 0 0.2825 Air/methane/CO₂ on BSA coated beads BLANK  1) BSAcontrol - distilled water collection 30 min 0.2795  2) BSA control -bead extraction 30 min 0.3145 5.87 Air/methane only MMO/ADH/FADHactivity only  3) Gas phase formic collected into 1 ml water 30 min0.3125  4) Beads extracted into water 30 min 1.3225 190.84  5) Gas phaseformic collected into 1 ml water 30 min 0.3145  6) Beads extracted intowater 30 min 1.3145 192.31 Air/methane/CO₂ MMO/ADH/FADH and FODHactivity  7) Gas phase formic collected into 1 ml water 30 min 0.269  8)Beads extracted into water 30 min 1.2775 366.08  9) Gas phase formiccollected into 1 ml water 30 min 0.2675 10) Beads extracted into water30 min 1.325 364.09

In addition to measuring the formate produced, the generation ofmethanol in the Mini-Column Reactor used above was also measured usingboth the liquid reagent and the methanol disk method outlined in Example1.

It is clear from FIG. 8, that methanol was produced by the MMOimmobilised onto the BSA coated silica beads, even in the presence ofall the other three enzymes (ADH, FADH and FODH).

This was quantified by the liquid methanol reagent as 86.9 μM in 30minutes. This value will be the amount of methanol in excess of thatbeing used by the ADH and indirectly the other two enzymes, as all fourenzymes were immobilised on the BSA silica beads and all were active asevidenced by the results obtained.

5. R-Propylene Oxide Production Using AMO

AMO cell free extract (500 μl˜3 mg enzyme) was added to 500 μl 100 mMMOPS pH 7.2 containing around 10 mg each of ADH.FADH and FODH thenthoroughly mixed with around 200 mg silica beads containing pre-driedBSA as an undercoat, then these were dried for 1.5 hours at 40° C. in avacuum oven over fresh silica gel. No extra NAD or NADH was added tothis first dried enzymes mixture. The dried beads/enzymes were ground toa fine powder in an agate mortar and pestle, then packed into 2 mm IDtubing (around 170 mg per tube) and assayed in the Climostat mini-rigsystem at room temperature using a mixture of air, methanol vapour andpropylene.

Propylene oxide generation was followed using a simple colorimetricassay system using the reaction N-benzyl pyridine with the propyleneoxide, which gives a bright purple colour when extracted into an organicsolvent layer (FIG. 8 on page 17).

AMO preparation μg in 30 minutes μg in 90 minutes AMO 1 — 11.62 AMO 26.38 11.92

The invention claimed is:
 1. A method of enzyme conversion, comprising:immobilizing an enzyme composition on a support material; drying thesupport material and enzyme composition to form a solid phaseimmobilized enzyme system with a water content of between 0.05% w/v to5% w/v; contacting the enzyme system with one or more reagents in thegas phase; allowing the enzyme system with a water content of between0.05% w/v to 5% w/v to convert the one or more reagent in gas phase toone or more product; wherein the enzyme composition is selected from thegroup consisting of a first enzyme plus a second enzyme, and multipleenzymes; and a co-factor convertable between first and second states;wherein the co-factor in the first state promotes reaction of the firstenzyme with a first gas phase reagent; and wherein the co-factor in thesecond state promotes reaction of the second enzyme with a second gasphase reagent.
 2. A method as claimed in claim 1, wherein the supportmaterial provides a micro-environment that maintains water around theimmobilised enzymes.
 3. A method as claimed in claim 1, wherein thesupport has a high internal surface area.
 4. A method as claimed inclaim 1, wherein the support material is a particulate support materialfluidised by passage of the gas phase reagent.
 5. A method as claimed inclaim 1, wherein the support material is an inorganic substrate with ahigh internal surface area.
 6. A method as claimed in claim 1, whereinthe co-factor is oxidized/reduced nicotinamide adenine dinucleotide(NAD/NADH).
 7. A method as claimed in claim 1, wherein the co-factor isoxidized/reduced flavin mononucleotide (FMN/FMNH).
 8. A method asclaimed in claim 1, wherein the co-factor is oxidized/reduced flavinadenine dinucleotide (FAD/FADH).
 9. A method as claimed in claim 1,wherein the co-factor is oxidized/reduced pyrroloquinoline quinone(PQQ/PQQH₂).
 10. A method as claimed in claim 1, wherein the firstenzyme is a monooxygenase enzyme.
 11. A method as claimed in claim 1,wherein the second enzyme or multiple enzymes is selected from the groupconsisting of: alcohol dehydrogenase, formaldehyde dehydrogenase,formate dehydrogenase, and a combination thereof.
 12. A method asclaimed in claim 1, wherein the one or more reagents comprises methaneand the product comprises methanol.
 13. A method as claimed in claim 1,wherein the one or more reagents comprises methanol and the productcomprises formic acid.
 14. A method as claimed in claim 1, wherein theco-factor oscillates between first and second states with multipleenzymes.
 15. An enzymic conversion system comprising: an enzymecomposition comprising a first enzyme, a second enzyme and a co-factorwhich may be converted between first and second states, immobilized on asupport material; wherein the co-factor in the first state promotesreaction of the first enzyme with a gas phase reagent; wherein theco-factor in the second state promotes reaction of the second enzymewith a gas phase reagent; and wherein the enzymic conversion system hasa water content of between 0.05% w/v to 5% w/v.
 16. An enzymicconversion system as claimed in claim 15, wherein the support materialprovides a micro-environment that maintains water around the immobilisedenzymes.
 17. An enzymic conversion system as claimed in claim 15,wherein the support has a high internal surface area.
 18. An enzymicconversion system as claimed in claim 15, wherein the support materialis a particulate support material fluidised by passage of the gas phasereagent.
 19. An enzymic conversion system as claimed in claim 15,wherein the support material is an inorganic substrate with a highinternal surface area.
 20. A sensor comprising a system as claimed inclaim 15.